Among the patents referenced above, the scattering cell described in U.S. Pat. No. 4,616,927, hereinafter referred to as the 927 flow cell or some of its slight modifications as shown in U.S. Pat. No. 5,404,217, hereinafter referred to as the 217 flow cell, has proven to be particularly useful for measuring the variation of scattered light intensity with scattering angle from particles in solution illuminated with a free light beam, such as produced by a laser. The term "particles" refers quite generally to the range of objects from macromolecules at the one extreme to large mammalian cells or even phytoplankton at the other. Characteristically, macromolecules whose molecular weights may be determined from their light scattering properties are of the order of 1000 gms/mol up to tens of millions of grams per mol. Their physical extent or size may vary from one nanometer to tens of micrometers. Prior to measuring the light scattered by the illuminated particles, it is very often desirable to separate them by chromatographic means such as size exclusion chromatography, or reverse phase chromatography. Connected on-line to such a chromatographic system and immediately following the separation columns, the 927 or 217 flow cell incorporated in a read head surrounded by an array of detectors provides a useful means for measuring the scattered light intensity from the separated sample flowing therethrough. From these measurements, the molecular weight and size distributions of the separated molecules may be determined following the standard techniques associated with these measurements. A detailed review and explanation of these methods, and how as they relate to the 927 flow cell, may be found in the 1993 article by P. J. Wyatt in Analytica Chemica Acta, volume 272, pages 1-40.
The 927 flow cell has been modified somewhat since its associated patent issued by changing its bore ends slightly and enclosing it into an integrated manifold structure such as disclosed in Design U.S. Pat. No. 329,821 by Wyatt and Shuck or, more recently, as disclosed in U.S. Pat. No. 5,404,217 by Janik and Magolske. Despite the many advantages of this type of flow cell for performing light scattering measurements on flowing samples following chromatographic separation, there are certain types of separations that produce exceptional sample resolution, yet function only for very small quantities of material. For example, when particles are separated by means of capillary hydrodynamic chromatography, or CHDF, as described, for example, in the article by Silebi and Dos Ramos in the Journal of Colloid and Interface Science, volume 130, pages 14 to 24, the capillary diameter is typically 60 .mu.m or less. The total sample volume injected into such a capillary would be of the order of 0.05 .mu.l. In order to measure the light scattering properties of this subsequently fractionated sample using the cell of the aforereferenced patents and co-pending application, the sample would have to be diluted by the square of the ratio of the flow cell bore diameter to the capillary diameter, or (1.25 mm/5.times.10.sup.-2 mm).sup.2 =625, since the flow cell bore is typically of the order of 1.25 mm. At this level of dilution after fractionation, not even considering the fractionation stage itself, which may dilute the injected volume by an order of magnitude or more, many fractionated samples will produce very low light scattering, hereinafter referred to by LS, signals or be undetectable. Indeed, the mismatch of flow channel diameters has appeared to be a major impediment to using the 927 flow cell to make light scattering measurements for samples eluting from such capillaries. Such huge volume dilutions have affected also the practicality of using refractive index detectors, with separations such as CHDF or capillary electrophoresis hereinafter referred to by CE. The similar small sample volume problems associated with CE are discussed, for example by Weinberger in his book Practical Capillary Electrophoresis.
Flow cytometry, hereinafter referred to as FC, represents another important area for the application of LS measurements. Because the illuminating laser sources are generally tightly focused, such as in the FC instruments manufactured for example, by Becton-Dickenson, Miles Laboratories, Ortho Diagnostics, or Coulter Electronics, it is particularly important to insure that the individual cells flow in a highly collimated and confined manner, as well. This type of flow constraint requirement is illustrated, for example, in U.S. Pat. No. 3,785,735 by Friedman et al. wherein is described a fine capillary based flow cell. Although some light scattering measurements are achieved by their design, the omnipresence of the various glass-liquid interfaces preclude great flexibility for making such measurements over a broad range of scattering angles. The design of the Friedman et al. flow cell insures very tight transverse control of the flowing particles by creating converging sheath flows all of which entrain the particles forcing them to flow through a fine capillary drilled into a larger glass structure. Once within the capillary itself, the entrained particles are expected to remain centered because of the occurance of laminar flow conditions. The main purpose of the sheath flow elements is to dilute and transport the particles into the narrow capillary in which they will be examined. The possibility that transverse spatial confinement could be achieved without the need for a stationary transparent capillary was not utilized by Friedman et al. nor to the designers of many types of so-called flow cytometers, including the types manufactured by the aforereferenced Coulter Electronics and Becton Dickenson companies.
In 1953, Crosland-Taylor reported in vol. 171 of Nature, pp 37-38, a new means for counting small particles. In this article he pointed out that narrow tubes, or capillaries, make " . . . microscopial observation of the contents of the tube[s] difficult due to the different refractive indices of the tube and the suspending fluid . . . " He also noted that such narrow tubes tend to block easily. Thus began his explanation of a device comprised of a relatively large tube through which flowed a stream of sheath-entrained particles. He described the sheath concept as comprised of the slow injection of a particle suspension into a faster stream of fluid flowing in the same direction. The faster sheath stream is, in turn, comprised of two sources from opposite sides of the injected sample. The two sources are needed to insure that the injected sample stream remains centered. For the Crosland-Taylor structure, particles are examined in regions where the diameter of the stream is the greatest. The particles are at a relatively great distance from the walls of the flow cell. For capillary-centered cells, on the other hand, the particles are very close to the flow cell boundaries. Conventional, flow cytometers of the types described earlier invariably rely on laminar flow containment and capillary flow cells of very small radius.
Microbore chromatography, a variant of liquid chromatography, is a separation technique which also involves the use of very small sample and solvent volumes. A sheath flow cell, of a simpler design than that of Crosland-Taylor used for detecting laser fluorescence in microbore chromatography has been reported by Hershberger, Callis, and Christian in Analytical Chemistry, volume 51, pages 1444 to 1446. Although the sample diameter and volume were as low as 0.05 mm and 6 .mu.l, respectively, the distance between the liquid/glass interfaces was about 10 mm, much larger than for flow cells used in the commercial flow cytometers discussed earlier. In this design, however, the liquid near the interfaces is essentially stationary and a single sheath contribution is provided by means of a flow within a tube concentric with the column exit flow stream.
The present invention, while providing a means for utilizing the 927 or 217 flow cell and avoiding the associated dilution problems, also presents a more general formulation for making light scattering measurements from a tightly confined sample.